Journal: bioRxiv

Article Title: Allosteric activation of CRISPR-Cas12a requires the concerted movement of the bridge helix and helix 1 of the RuvC II domain

doi: 10.1101/2022.03.15.484427

Figure Lengend Snippet: Structural organization of Francisella novicida Cas12a (FnCas12a) and influence of helix 1 mutations on the overall structure and thermo stability. ( A ) Domain organization of FnCas12a with mutation sites indicated in red. ( B ) Structure of the binary complex of FnCas12a with crRNA (orange) (PDB: 5NG6). ( C ) Structure of the ternary complex of FnCas12a together with crRNA (orange) and target DNA (black) (PDB: 6I1K). ( B ) and ( C ) Protein domains are colored according to the domain organization in (A). Helix 1, a part of RuvC II domain, is colored in dark blue, the bridge helix (BH) in green. ( D ) and ( E ) Close-up of helix 1 (blue) and BH (green) in the binary (D) and ternary (E) state, showing the change of helical lengths upon the rearrangement from binary to ternary complex. Mutation sites I960, K978, and K981 are shown in red. ( F ) Far-UV CD spectra of WT FnCas12a and FnCas12a helix 1 variants (2 µM) at 25 °C. Shown is the average of five replicates. ( G ) Protein Thermal Shift™ (Thermo Scientific) melting curves of FnCas12a variants (2 µg) from 25 to 95 °C. Shown is the average of four replicates.

Article Snippet: The matched target MM 0 as well as the mismatched target DNA sequences (MM 2, MM 10, MM 20), were purchased from Eurofins-MWG (Ebersberg, Germany, Table S2 ).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Allosteric activation of CRISPR-Cas12a requires the concerted movement of the bridge helix and helix 1 of the RuvC II domain

doi: 10.1101/2022.03.15.484427

Figure Lengend Snippet: Single molecule Förster resonance energy transfer (FRET) measurements of freely diffusing molecules were conducted of doubly labeled FnCas12a REC-Nuc*DL550/DL650 helix 1 variants in comparison to WT FnCas12a REC-Nuc*DL550/DL650 . ( A ) Schematic illustration of FnCas12a with labeling positions in the Nuc domain (T1222) and the REC domain (D470). ( B ) FRET efficiency histograms of FnCas12a I960P/K981P REC-Nuc*DL550/DL650 , FnCas12a Δh1 REC-Nuc*DL550/DL650 , and FnCas12a ΔBH/Δh1REC-Nuc*DL550/DL650 compared to WT FnCas12a REC-Nuc*DL550/DL650 of the apo enzyme, the binary complex (1 nM crRNA), the ternary complex (1 nM crRNA, 1 nM target DNA), and the ternary complex without NTS (1 nM crRNA, 1 nM TS DNA). Fitting of the histograms was performed with a double or triple Gaussian function. The averaged mean FRET efficiencies (E) and the percentage distribution of the populations (A) are given with SEs in the histograms. Measurements were performed in triplicates (see also Supplementary Figure S8).

Article Snippet: The matched target MM 0 as well as the mismatched target DNA sequences (MM 2, MM 10, MM 20), were purchased from Eurofins-MWG (Ebersberg, Germany, Table S2 ).

Techniques: Förster Resonance Energy Transfer, Labeling

Journal: bioRxiv

Article Title: Allosteric activation of CRISPR-Cas12a requires the concerted movement of the bridge helix and helix 1 of the RuvC II domain

doi: 10.1101/2022.03.15.484427

Figure Lengend Snippet: Plasmid cleavage assay of FnCas12a h1 variants. The binary complex (37.5 nM FnCas12a + crRNA) was incubated with 5 nM of target DNA at 37 °C for 1 h. ( A ) The linearized target DNA plasmid (7300 bp) is cleaved into two products (4400 and 2900 bp) and analyzed on a 0.8% agarose 1x TAE gel. ( B ) The supercoiled plasmid DNA is nicked or linearized by FnCas12a variants and analyzed on a 1.5% agarose 1x TAE gel. 1 kb plus DNA ladder.

Article Snippet: The matched target MM 0 as well as the mismatched target DNA sequences (MM 2, MM 10, MM 20), were purchased from Eurofins-MWG (Ebersberg, Germany, Table S2 ).

Techniques: Plasmid Preparation, Cleavage Assay, Incubation

Journal: bioRxiv

Article Title: Allosteric activation of CRISPR-Cas12a requires the concerted movement of the bridge helix and helix 1 of the RuvC II domain

doi: 10.1101/2022.03.15.484427

Figure Lengend Snippet: High-resolution cleavage assay of FnCas12a helix 1 variants in comparison to FnCas12a BH variants. The 58 nt double stranded target DNA carries a Cy5 label at the TS and a Cy3 label at the NTS. A 7.5-fold excess of binary complex (Cas12a and crRNA, 750 nM) was used over the target DNA (100 nM). The reaction was incubated for 1 h at 37 °C. ( A ) DNA strands are labeled with Cy3 (green sphere) and Cy5 (red sphere) fluorophores at the 5’-end of the DNA strands, respectively. ( B ) DNA strands are labeled with Cy3 (green sphere) and Cy5 (red sphere) fluorophores at the 3’-end of the DNA strands, respectively. ( C ) Constructs shown in (A) were used. In addition to the previously reported shift of NTS cleavage products of the BH variants the helix 1 deletions variants (Δh1, ΔBH/Δh1) show a shifted cleavage pattern for the TS. ( D ) Constructs shown in (B) were used. The shift in TS cleavage for FnCas12a Δh1 and FnCas12a ΔBH/Δh1 is again observed. FnCas12a I960P/K981P does not show cleavage at all. Samples were analyzed on a denaturing 15% PAA gel.

Article Snippet: The matched target MM 0 as well as the mismatched target DNA sequences (MM 2, MM 10, MM 20), were purchased from Eurofins-MWG (Ebersberg, Germany, Table S2 ).

Techniques: Cleavage Assay, Incubation, Labeling, Construct

Journal: bioRxiv

Article Title: Allosteric activation of CRISPR-Cas12a requires the concerted movement of the bridge helix and helix 1 of the RuvC II domain

doi: 10.1101/2022.03.15.484427

Figure Lengend Snippet: Mechanistic model summarizing the impact of helix 1 on FnCas12a activity. ( A ) Loading of target DNA leads to the formation of the ternary complex. This transition is accompanied by an opening of the enzyme and a tandem movement of helix 1 and the bridge helix (BH), e.g. a shortening of helix 1 (blue cylinder) and extension of the BH (green cylinder). Additionally, the lid domain (cyan) is restructured inducing the formation of a short helix and ultimately the opening and activation of the active site in the RuvC II domain. ( B ) Introduction of proline residues in helix 1 and the BH (kink in the cylinders), respectively, prevents the concerted tandem movement of helix 1 and the BH. The impaired movement of helix 1 is translated to the lid domain that cannot reorder ultimately preventing the opening of the lid. Consequently, the active site is not activated and cis - and trans -DNA cleavage activity cannot be observed.

Article Snippet: The matched target MM 0 as well as the mismatched target DNA sequences (MM 2, MM 10, MM 20), were purchased from Eurofins-MWG (Ebersberg, Germany, Table S2 ).

Techniques: Activity Assay, Activation Assay